Application of a green fluorescent fusion protein to study protein-proteininteractions by electrophoretic methods

A screening procedure for protein-protein interactions in cellular extracts using a green fluorescent protein (GFP) and affinity capillary electrophor esis (ACE) was established. GFP was fused as a fluorescent indicator to the C-terminus of a cyclophilin (rDmCyp20) from Drosophila melanogaster Cyclop hilins (Cyps) belong to the ubiquitously distributed enzyme family of pepti dyl-prolyl cis/trans isomerases (PPlases) and are well known as cellular ta rgets of the immunosuppressive drug cyclosporin A (CsA). The PPlase activit y of the GFP fused rDmCyp20 as well as the high affinity to CsA remain inta ct. Using native gel electrophoresis and ACE mobility-shift assays, it was demonstrated that the known moderate affinity of Cyp20 to the capsid protei n p24 of HIV-1 was detectable in the case of rDmCyp20 fused to the fluoresc ent tag. For the p24/rDmCyp20-GFP binding an ACE method was established whi ch allowed to determine a dissociation constant of K-d = 20 +/- 1.5 x 10(-6 ) M. This result was verified by size-exclusion chromatography and is in go od agreement with published data for the nonfused protein. Moreover the fus ion protein was utilized to screen rDmCyp20-protein interactions by capilla ry electrophoresis in biological matrices. A putative ligand of rDmCyp20 in crude extracts of embryonic D. melanogaster was discovered by mobility-shi ft assays using native gel electrophoresis with fluorescence imaging and AC E with laser-induced fluorescence detection. The approach seems applicable to a wide range of proteins and offers new opportunities to screen for mode rate protein-protein interactions in biological samples.